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Published in final edited form as: Chem Biol Interact. 2020 May 11;325:109132. doi: 10.1016/j.cbi.2020.109132

Interaction of alcohol with time of eating on markers of circadian dyssynchrony and colon tissue injury

Faraz Bishehsari 1,*,€, Fabian Preuss 2,*, Seyed Sina Mirbagheri 1, Lijuan Zhang 1, Maliha Shaikh 1, Ali Keshavarzian 1,3,4,5
PMCID: PMC7315934  NIHMSID: NIHMS1595802  PMID: 32437693

Abstract

Background:

Alcohol increases the risk of developing colon cancer (CRC), in part via tissue inflammation and impaired barrier integrity. Circadian dyssynchrony (CD) is an understudied but common lifestyle associated factor that increases the risk of multi-organ tissue injury and number of malignancies including CRC. Our prior studies showed that the shift in light-dark cycle exacerbates barrier dysfunction and colonic inflammation in the setting of alcohol treatment, and increases the risk of CRC. Here we studied the interaction of alcohol with an abnormal eating pattern on markers of CD and colonic barrier integrity.

Method:

Mice were subjected to day (rest-phase=wrong-time WT) or night-time (active-phase=right-time RT) access to food in combination with access to water or 15% alcohol for total duration of 10 weeks. The food and liquid intake was measured. The locomotor activity data was recorded throughout the study, using a beam-break system. Mice were euthanized at two time points (ZT2 and ZT14). Time variation in the expression of the molecular marker of circadian clock (per2 gene) was measured in the central (hypothalamus) and intestinal (colon) tissue. Colonic protein expression of barrier markers (Occludin and Claudin-1) was studied.

Results:

No significant differences were present in the weight gain and alcohol intake among the groups over the study period. We observed an interaction of WT eating with alcohol on behavioral markers of circadian rhythm. Compared to the RT+ Water treated animals (“reference group”), combination of WT eating and alcohol consumption (WT+Alcohol) significantly changed the per2 oscillatory pattern, that was different between the colon and hypothalamus, indicative of worsening circadian dyssynchrony. This was associated with an overall impaired expression of barrier integrity markers in the colon.

Conclusions:

Alcohol induces circadian dyssynchrony which is worsened by abnormal food timing, associated with impaired barrier integrity in the colon. Future studies on the interaction of alcohol and food timing could provide further insights into alcohol associated CRC pathophysiology.

Keywords: alcohol, circadian, food timing, barrier, colon carcinogenesis

Colorectal cancer (CRC) is among the top three most common cancers and most common causes of cancer related death in the world (Center et al., 2009, Bishehsari et al., 2014). While only a small portion (<5%) of CRCs are caused by known genetic predisposition, most CRC cases occur sporadically without a known genetic background (Migliore et al., 2011). In fact, epidemiological studies confirm the strong role of environmental factors on modulating the risk of CRC (Lochhead et al., 2014). Among such factors, alcohol consumption is a well-established risk factor for CRC (Fedirko et al., 2011). Identifying mechanisms and co-factors that may accelerate alcohol induced colon carcinogenesis could help us with risk stratification and preventive strategies to reduce the burden of the disease (Ma and Ladabaum, 2014).

CRC arises from multiple genetic and epigenetic abnormalities that accumulate within a permissive chronic inflammation background leading to tumor formation (Rossi et al., 2018b). While it has been known for a long time that chronic inflammation increases the risk of CRC in patients with inflammatory bowel disease, the link between inflammation and tumorigenesis in sporadic CRC has only recently been recognized (Moossavi and Bishehsari, 2012). Prior studies have shown that inflammation is a feature of alcohol associated colon carcinogenesis (Wimberly et al., 2013, Shukla et al., 2016). Intestinal inflammation is indeed a prominent feature associated with alcohol consumption, based on animal as well as human studies(Bishehsari, 2016). Chronic alcohol consumption could impair gut barrier integrity, an early event in intestinal inflammation (Parlesak et al., 2000). In addition to the direct effect of alcohol on the epithelial cells, alcohol can also impair mucosal integrity via disruption of the transepithelial space, consisting of tight junction proteins (Wang et al., 2014, Keshavarzian et al., 2009). Barrier dysfunction, an indicator of subclinical inflammation, is an early event in colon carcinogenesis (Bongers et al., 2014, Grivennikov et al., 2012).

Circadian dyssynchrony (CD) is another environmental factor associated with our modern lifestyle habits (Bishehsari et al., 2016a). Varieties of biological processes including immune components are under circadian control (Scheiermann et al., 2013, Granda et al., 2005); thus, predisposition to intestinal inflammation from CD doesn’t seem to be unexpected. In fact, light-dark shift schedule, a common form of CD, exacerbates gut barrier dysfunction and colonic inflammation (Preuss et al., 2008) and increases the risk of CRC (Schernhammer et al., 2003).

Interestingly, recent data has shed light on the interaction of alcohol and CD on intestinal homeostasis. CD from light-dark shift could amplify alcohol-induced intestinal permeability and inflammation (Summa et al., 2013). More recently, we showed that Light-dark shifting exacerbates alcohol-induced polyposis and CRC, an effect that could be mediated via a pro- tumorigenic inflammatory milieu (Bishehsari et al., 2016b). While CD could promote alcohol- induced gut barrier integrity and inflammation, alcohol itself may cause disturbances in the circadian rhythm; In individuals who consume alcohol, impairment of the intestinal barrier integrity is associated with low levels of melatonin, a marker for disrupted central circadian rhythm (Swanson et al., 2016). However, the effect of alcohol on the colonic circadian homeostasis has not been studied yet.

While light-dark shifts have been widely used to model CD, abnormal eating patterns enforced by food restriction, defined as eating close or during the physiologic rest time, is an under-studied but common form of CD (Wehrens et al., 2017). Restricting feeding at rest (“wrong time”) can shift the circadian oscillation of the colon clocks, leading to CD (Stokkan et al., 2001, Hoogerwerf et al., 2007).

Here we examined the effect of wrong time eating on alcohol associated CD with studying peripheral and central circadian genes’ differential expression. Effect of the CD on colonic barrier integrity was measured.

Methods:

Animal housing and food / water / alcohol consumption:

C57BL/6J, male mice starting at 8 weeks of age, housed in a light and temperature controlled environment at a conventional animal facility were used for this study. The mice were subjected to a food restricted paradigm, with either day (rest-phase=”wrong-time”) or night-time (active-phase=”right-time”) access to food for 12 hours for 5 consecutive days followed by a 2-day ad-libitum access to food, modeling weekend ad-lib in humans. This paradigm was repeated for a total duration of 10 weeks. Alcohol treatment: Subsets of animals (n=5 each) were given water or 15% ethanol alcohol during their food access phase, followed by regular water during the food restricted phase. Time is expressed as Zeitgeber time (ZT), with ZT12 defined as the time of lights off in LD conditions. All animals were singly housed, locomotor activity was measured using a beam-break system with three beams penetrating the cage counting # of beam breaks per minute, and each animal had 2 separate housing cages (one with and one without food). The mice were transferred during the light-transition phases (ZT0 & ZT12) to enter or leave the food home cage. Food and Liquid consumption were measured at the end of each 5 day restricted period when the mice were leaving their food containing home cage (ZT0 & 12 respectively) and right after the weekend during their first starvation period. At the end of the study, mice of each group were euthanized at ZT2 and ZT14 (n=2–3 per ZT) and the colon and the hypothalamus were collected for gene expression and protein analysis as detailed below.

During the first week, the food and ethanol supplementation was graduated, beginning with 6, 9 & 12 hours of food restriction combined with 5%, 10% and 15% ethanol supplementations spread across 2+2+1 day increments to ease the animals’ adaptation, followed by the first weekend setting.

The study method was approved by the Institutional Animal Care and Use Committee of Rush University Medical Center.

Recording locomotor activity:

The animals were singly housed in triple beam break system equipped cages and monitored for a total duration of 10 weeks. The locomotor activity data was recorded and analyzed using the Chronobiology Kit from Stanford Software Systems. The number of beam breaks per 1 minute was recorded in 1 minute bins and total counts calculated to determine activity patterns during the various activity periods. Locomotor activity data from the first week has been omitted in the analysis due to the graduate enforcement of food restriction. The day-time versus night time overall activity was calculated by restricting the periods to 10 hour blocks ZT2-ZT10 (lights on) and ZT14-ZT22, omitting 2 periods of 4 hour transition time. The increased locomotor activity in these transition periods was induced due to the required cage change either adding or removing the food and omnipresent in all groups causing a novelty effect throughout the study.

RNA:

Animals were euthanized via cone-assisted decapitation to ensure minimal effects of the euthanasia on RNA expression profiles. The whole brain was removed and the hypothalamus dissected out and immediately placed in RNA-later solution for RNA extraction. Similarly, the proximal colon was immediately dissected and placed in RNA-later. RNA was extracted using the RNeasy Mini RNA Extraction Kit (Qiagen, Hilden, Germany). cDNA was prepared using the high capacity cDNA reverse transcription kit from the manufacturer (Applied Biosystems, Foster City, CA).

The real time PCR was performed on an Applied Biosystems 7900HT Fast apparatus using per2 primers (forward: 5′- CTC CAG CGG AAA CGA GAA CTG - 3′; reverse: 5′- TTG GCA GAC TGC TCA CTA CTG - 3′) and Fast Sybr green (Applied Biosystems). The quantitative analysis was calculated from the ΔΔ^ values and normalized against the β-actin (forward: 5′-GTG ACG TTG ACA TCC GTA AAG A −3′; reverse: 5′-GCC GGA CTC ATC GTA CTC C −3′) as housekeeping.

Protein:

Protein concentration was quantified and samples prepared as follows: 20ug total protein plus 20ul of Laemmli sample buffer (Bio-RAD). Samples were boiled for 5 minutes and loaded in each lane to 10% SDS gel. Samples were run at 100V for 2 hours and transferred to the Nitrocellulose blotting Membrane (GE Healthcare life Sciences CAT#10600004), and blocked with 5% w/v nonfat dry milk for 1 hour. RT. After washing with TBST, membranes were incubated overnight at 4 °C with primary antibodies Occludin (mouse) (Invitrogen #33–1500) and Claudin-1(rabbit) (Invitrogen # 51–9000) on the gentle shaking. The membranes were further incubated with corresponding HRP conjugated secondary antibodies for 1 h at room temperature and revealed by ECL solution. For quantification, we used the Image J program, after normalization to expression of actin (mouse; Sigma#A4700).

Statistics:

Data are presented as mean ± STDM. The effects of alcohol and/or food timing and locomotor activity were calculated using two-factor ANOVA. P values of 0.05 or less were considered to be statistically significant.

Results:

Food and alcohol consumption

Average food intake during the weekdays was decreased both by feeding time (F(10.509),p=005), as well as the alcohol treatment (F(8.268),p=0.011), with mice fed during the wrong time and supplemented with alcohol (WT+ Alcohol) showing the lowest food intake during the weekdays. The WT feeding groups, however, showed a rebound in food intake during the weekend ad-lib, showing a significantly large increase in food consumption during the 2-day continuous food availability period (RT 4.7g±0.5g; WT 5.4g±0.7g; RT+ Alcohol 4.7g±0.5g; WT+ Alcohol 5.3g±0.3g), with a significant effect of WT feeding time, and not alcohol, on the weekend food consumption (Feeding time, F15.864,p=0.001). The weekend food intake rebound seemed to be sufficient to compensate for the differential food intake between the weekdays and weekends as no significant differences in the final bodyweight and overall weight gain was observed among groups over the study period (Figure 1a).

Figure 1 :

Figure 1 :

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Feeding time interacts with alcohol intake on the behavioral marker of circadian rhythm: a) Final bodyweight was not different among the studied groups. b) Representative actogram of locomotor data for the Right time+Water fed ( “Reference+Water”) group, showing a nocturnal activity predominantly, that became more robust during the ad-lib weekends (red boxes), with minimal changes in the activity patterns (e.g., recovery) when transitioning from weekends to weekday (green boxes) periods. Transition times (ZT0 and ZT12) showed a strong locomotor activity. The table shows the average activity data (with the standard deviation/SD) during the restricted periods (weekdays) versus ad-lib weekends per treatment (n=5/group).

The average water intake during weekdays was also affected by feeding time and alcohol intake (Two-way ANOVA: Time of feeding (F(79.606),p=0.000); alcohol F(7.651),p=0.014); Interaction F(6.162), p=0.025). During the weekends, there was a modest effect of WT eating and alcohol interaction on the water intake (Interaction F(4.669),p=0.046). No significant differences in the alcohol consumption were observed between WT and RT eating groups.

Locomotor activity

The animals’ locomotor activities, as the established behavioral marker of circadian timekeeping, were monitored during the study. The activity of the animals was monitored using the Stanford Software Systems Chronobiology Kit throughout the entire duration of the study. In the final analysis, the first week data was omitted due to the gradual introduction of the food restriction and the consequent disruption of the animals’ behavior. A strong anticipatory behavior was observed in all groups prior to the semi-daily cage change, granting or restricting the access to food with or without alcohol. All groups showed strong locomotor activities around these transition times (ZT0 or ZT12) with no differences among them. We therefore focused our analysis on the day or night time periods starting two hours after the last and two hours prior the next transition phases: day ZT2-ZT10, night ZT14-ZT22.

We observed significant effects of feeding time and alcohol on the locomotor activity during the day food restriction paradigm as well as carry-over effects from the restricted periods into the unrestricted food access locomotion during the weekend ad-lib (Figure 2b). The night periods remained with the highest amount of total activity in all groups despite the opposite feeding paradigms. The daytime activity was significantly increased in WT+ Alcohol animals, showing an interaction of WT eating and alcohol (two-way ANOVA; Interaction F(4.895),p=0.043).

Figure 2:

Figure 2:

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WT eating worsens the alcohol induced changes in the molecular marker of circadian rhythms: a) Differential expression of per2 across time in the central (hypothalamus) and b) intestinal (colon) tissue in Reference (right time fed) water treated group, versus alcohol treated groups combined with right-time (RT) or wrong-time (WT) feeding. Error bars represent mean ± SE (3 mice per ZT). (*) indicates p < 0.05. ZT, Zeitgeber. c) Average ZT2 to ZT14 ratio of per2 per site per group relative to the Reference group.

During the ad-lib weekend periods, we made two observations; compared to the RT fed groups, which showed a strong nocturnal behavior (F(11,863),p=0.004), WT groups did not decrease their daytime activity while food was available throughout the day and alcohol reduced the overall nocturnal activity (F(4.550),p=0.05). WT fed animals needed a recovery phase from a predominant nocturnal behavior, when transitioning from the weekends to weekday periods, with the WT+ Alcohol group showing the slowest recovery in the weekend to weekday transition.

Overall, this data suggests an interaction of WT eating with alcohol on behavioral markers of circadian rhythm. Next, we directly tested such an interaction on the molecular markers of circadian rhythm in central (hypothalamus) and intestinal (colon) tissues.

Alcohol combined with wrong time eating affects the oscillation of per2 gene expression in the central and intestinal tissue:

We examined the effect of WT eating on the alcohol’s impact on the differential expression of per2 gene between two ZTs during the light (ZT2) or dark phase (ZT14) in the hypothalamus of the studied mice. Compared to the “Reference” group (RT water treated) animals, the brain ZT2 to ZT14 ratio of per2 gene expression was reduced, though non-significantly, in mice treated with alcohol combined with RT eating (Figure 2a ), an effect that became significant only when alcohol was combined with WT eating (p=0.036).

Similarly, combination of alcohol and WT resulted in a significant change in the ZT2 to ZT14 ratio of per2 gene in the colon compared to water treated animals (Figure 2b; p=0.027). However, the direction of the changes imposed by alcohol and WT eating in the per2 differential expression across the ZTs appeared to be divergent in the hypothalamus and colon (Figure 2c), suggesting a central-peripheral circadian dyssynchrony.

Alcohol combined with wrong time eating induces circadian dyssynchrony:

In order to directly examine the circadian dyssynchrony, we estimated the molecular markers of central-peripheral circadian clock by calculating the brain to colon (BC) ratio of per2 expression at each time point, and across the time. The reference group showed a higher ratio of BC at ZT2 to BC at ZT14, a pattern that was reversed by alcohol treatment, and overall was suppressed when alcohol was combined with WT eating (Figure 3a). Compared to the controls, the mean ratio of BC at ZT2 over BC at ZT14 dropped remarkably in the WT+ Alcohol group (Figure 3b). Two-way ANOVA confirmed a significant effect of alcohol (p=0.001), food timing (p=0.001), as well as their interaction (p=0.002) on the time variation in the per2 expression in the hypothalamus relative to that in the colon.

Figure 3:

Figure 3:

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Alcohol combined with WT eating induces circadian dyssynchrony: a) The trend of brain to colon (BC) ratio of per2 expression at each time point per group, b) ZT2 to ZT14 average ratio of brain to colon (BC) per2 expression per treatment (n=5/group).

Alcohol combined with wrong time eating impairs colonic barrier markers:

Impaired epithelial barrier precedes colon tumor formation and occurs early during the colon carcinogenesis. (Soler et al., 1999, Puppa et al., 2011, Grivennikov et al., 2012). We examined whether circadian dyssynchrony, induced by alcohol and WT eating could be associated with changes in the colon expression of tight junction proteins, biomarker of barrier integrity.

Alcohol combined with WT eating tended to have a decreased expression of Occludin in the colon, compared to control water fed animals (Figure4a, P=0.051). Similarly, Claudin-1 expression reduced in the WT+ Alcohol group (Figure4b, P=0.069). Average changes on the tested barrier markers as an estimation of the overall barrier integrity revealed a significant reduction in the colonic barrier markers in the WT+ Alcohol group (Figure4c, p=0.026).

Figure 4:

Figure 4:

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Alcohol combined with WT eating impairs markers of colonic barrier integrity: a) Occludin and b) Claudin-1 expression was normalized to Actin; Representative graph of the expression of barrier markers are shown; c) Percent changes on the Occludin and Claudin-1 expressions relative to the control, water treated (Reference Water) group. Bar graphs show means ± SE, n=5/group, asterisk indicates p<0.05.

Discussion:

Colon cancer is the most common gastrointestinal cancer worldwide. Pathogenesis of CRC is linked to lifestyle related risk factors including alcohol consumption (Rattray et al., 2017). We and others showed that the underlying mechanism of alcohol associated CRC is in part via the induction of intestinal inflammation and impaired barrier integrity (Wimberly et al., 2013, Shukla et al., 2016) , a phenomenon that is exacerbated by light-dark shift, a well-studied form of circadian dyssynchrony (CD)(Bishehsari F, 2017). Here we show that alcohol consumption by itself induces differential expression of the peripheral and central circadian gene, indicative of CD. Wrong time eating, a common yet understudied form of CD, exacerbated alcohol induced-CD, which was associated with an increased impairment of intestinal barrier integrity markers.

Accumulating data suggests a bidirectional link between alcohol induced organ damage and circadian rhythms (Udoh et al., 2015, Chen et al., 2004). Alcohol-induced gut leakiness and the subsequent alcoholic liver disease is enhanced when circadian rhythm is disrupted either by genetic or environmental (light-dark shift) modes of circadian disruption (Summa et al., 2013). In our study, alcohol consumption alone significantly affected the colonic and not central differential expression of per2 gene. This is consistent with prior reports showing the differential effect of alcohol on rhythmic expression of per genes in the central versus peripheral (liver) organs (Chen et al., 2004, Filiano et al., 2013, Zhou et al., 2014), and that gene expression responses to chronic alcohol exposure are tissue-specific (Summa et al., 2015). Interestingly, wrong time eating aggravated the alcohol’s effect on the differential expression of the per2 gene in both sides, causing an enhanced CD in our model.

Both genetic as well as environmental modes of CD have been previously linked to colonic barrier dysfunction, inflammation and carcinogenesis(Bishehsari et al., 2016a). The impaired expression of barrier markers in the WT+ Alcohol treated animals suggests that changes induced by CD may contribute to pathological changes of the early steps of CRC.

The mechanisms underlying the susceptibility of the clock machinery to chronic alcohol consumption is unclear and probably different between the central and peripheral clocks. It has been proposed that the alcohol’s effect on the hepatic clock could be due to the NAD(H) dependent regulation of DNA binding of the clock to E-box elements(Udoh et al., 2015). It is noteworthy that the alcohol metabolism in the liver is predominantly via dehydrogenases, resulting in alterations in NAD+ /NADH ratio, while the dehydrogenase activity in the colon is relatively low (Na and Lee, 2017). Besides dehydrogenation, acetaldehyde, the major metabolite of alcohol in the colon, mainly forms upon bacterial metabolism in the colon (Seitz et al., 1990, Seitz and Stickel, 2010, Rossi et al., 2018a). The colon is home to a large number of bacteria that could affect the host gene expression either directly or via their metabolites (Kelly et al., 2015, Tahara et al., 2018). Alcohol has been shown to alter the gut microbiota both in human and animals (Engen et al., 2015). On the other hand, a disrupted clock either in genetic models or via environmental cues (light-dark shift or abnormal eating pattern) also alters the gut microbiota (Voigt et al., 2014, Rosselot et al., 2016). It is possible that alcohol via CD or other mechanisms alter microbiota, which could then impact barrier integrity and colonic inflammation. The former notion is supported by our observation that worsening CD by wrong time eating in the alcohol treated mice was associated with decreased expressions of barrier markers. Nevertheless, we have not interrogated microbiota in our study.

The effect of WT eating on the worsening alcohol-induced CD can be explained by the effect of food on colonic circadian rhythm. In the liver, another metabolically active peripheral digestive organ, restricted time of feeding shifts the liver’s clock from the central clock, causing a central-peripheral CD (Udoh et al., 2015). A similar phenomenon could explain the promoting effect of WT on the alcohol induced central-colonic CD in our model.

Thus, our study shows for the first time that abnormal food timing could enhance alcohol induced circadian dyssynchrony, which could be translated into impaired barrier integrity, an early step in colonic inflammation and carcinogenesis, and thereby provides insights into alcohol associated CRC pathophysiology. Further studies are needed to determine the mechanisms of the interaction between alcohol and food timing, and whether the observed molecular alterations could contribute to the CRC burden.

Acknowledgments:

The authors are grateful to Marissa K Greathouse for her technical assistance in animal care.

Funding: Faraz Bishehsari is supported by NIH/NIAAA: AA025387 as well as Rush Translational Sciences Consortium/Swim Across America Organization grant. Ali Keshavarzian is supported by NIH/NIAAA: AA023417.

Abbreviations:

CRC

colon cancer

CD

circadian dyssynchrony

PER

Period

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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